MiSeqs with MCS 2.6 or earlier include several pre-installed
reference genomes that are used during the alignment and variant
calling steps of many MiSeq
Reporter analysis workflows, which can also utilize user-added
custom reference genomes. A custom reference genome file must be
properly formatted and added to the MiSeq Reporter and Illumina
Experiment Manager Genome repositories so that it can be used
for analysis in MiSeq Reporter. Custom reference genomes cannot be
used for analysis in BaseSpace.
- The file must be in FASTA format using the *.fa extension, not
the *.fasta extension.
- Use a text editor such as Notepad or
TextEdit, or a source code editor like Notepad++, to edit your
- Sequence identifiers are limited to 24 characters and
can only contain letters, numbers, hyphens, and underscores. All
other characters, including blank spaces, are not allowed in the
- Examples of good and bad sequence
- For the
sequences, each line in the file is limited to 80 characters in
length. The sequences must contain only the characters A, T, C and G
and may use only upper-case characters. Ns, blank spaces, and lower
case characters are not allowed.
- Each line in the file must
end with a line feed character (LF), not a carriage return (CR), nor
both a CR and LF. Some source code editors such as Notepad++ include
an option to view all characters, which displays invisible
characters such as end of line characters. In Notepad++, to change
all end of line characters to LF:
- Go to Edit >
EOL Conversion > Unix Format.
Add your reference file to the MiSeq Reporter Genome repository:
- On the MiSeq PC, navigate to the Genome repository. The
default path is C:\Illumina\MiSeq Reporter\Genomes.
- In the
Genomes folder (repository), create a new folder and give the new
folder a genome name. Avoid using any spaces in the name.
- Place your custom reference genome FASTA file inside the new
Add your reference file to the Illumina Experiment Manager (IEM)
- Open IEM and select the Folder Settings button. Take
note of the Genome Repository path.
- Navigate to the
Genomes folder shown in the Genome Repository path.
- Note: The default Genomes directory is often hidden by
default. Either copy and paste the path from the folder settings
to a new Computer window or configure Windows to view hidden
files in order to follow the full path. To view hidden files,
open the Windows Control Panel, select Folder
Options, and click on the View tab. Select Show
hidden files, folders and drives and uncheck the Hide
extensions for known file types setting.
- In the Genomes folder, create a new folder and give the
folder a genome name (do not use spaces in the name), then place
your custom reference genome FASTA file inside the new folder.
- Close and re-open IEM.